Project Summary The community of microorganisms within our gastrointestinal tract, collectively known as the gut microbiota, constitutes one of the densest and most diverse bacterial ecosystems known. While the close relationship between humans and their microbiota represents vast potential for engineering human health, we are currently limited in tools required to unravel the intrinsic complexity. Our ability to predictably harness the microbiota for beneficial health outcomes requires a fundamental understanding of the physiology of these bacteria, yet most human gut bacteria have never been studied using molecular genetic tools and are too distantly related from well-studied model bacteria to accurately transfer gene annotations by homology. This major gap in our functional understanding of gene functions in human gut bacteria must be addressed with systematic efforts, which will require multiple complementary expertise. High-throughput genetics is an attractive approach for characterizing the biological functions of genes within the human microbiota. Application of perturbations en masse to large populations of genetically modified bacteria permits the parallel assessment of nearly all genes. A similar high-throughput strategy can potentially be applied to the human gut microbiome, but there are multiple major obstacles that we aim to resolve in this project: (1) transformation of non-model bacteria remains challenging and is a largely trial-by-error effort, (2) the development of a new genetic system for a non-model bacterium is time-consuming, (3) the adoption of multiple technologies and laboratory workflows complicates the comparison of data across teams, (4) in vivo mouse experiments should ideally be carried out in ex-germ-free mice colonized by mutants of interest. The team assembled for this grant includes leaders at the forefront of novel cultivation methods, electroporation for genetic transformation, and tools for assessing gene function in vitro and in vivo. In Aim 1, we will rapidly develop genetic tools for a large number of human gut commensal strains, with the ultimate goal of generating genome-wide randomly barcoded transposon mutant libraries for sequencing. We will utilize these libraries to test the phenotypic importance of all non-essential genes across a multitude of in vitro (Aim 2) and in vivo (Aim 3) conditions to globally discover new gene functions. Through our combined expertise in bacteriology, microfluidics, high-throughput screening, host-microbe interactions, and imaging, we will produce genetic tools and fitness data for the vast community of microbiota researchers at unprecedented scale, and deliver deep insight into the physiology of the human gut microbiota.